Systems, Methods, And Compositions For The Rapid Early-Detection of Host RNA Biomarkers of Infection And Early Identification of COVID-19 Coronavirus Infection in Humans

Abstract

The current inventive technology is directed to systems, methods, and compositions detection of host signatures of pathogenic infection, and in particular a rapid detection assay configured to detect target RNA transcripts that may be biomarkers of infection. In one embodiment, the invention includes systems, methods and compositions for the early detection of pathogens or infection in an asymptomatic subject through a novel lateral flow assay, which in a preferred embodiment may include a rapid self-administered test strip configured to detect one or more RNA transcript biomarkers produced by a subject's innate immune system in response to a pathogen or infection and present in saliva.

What is claimed is: 
     
         1 . A method of detecting a host RNA transcript biomarker comprising the step of:
 collecting a bodily fluid sample from a subject containing an RNA transcript biomarker;   converting said RNA transcript biomarker into a DNA probe, such as a double stranded DNA (dsDNA), single stranded DNA (ssDNA), or and a hybrid double stranded DNA (dsDNA) probe having:
 a dsDNA target sequence; 
 a single stranded DNA (ssDNA) annealing region; and 
 a ssDNA target capture region; 
   introducing said hybrid dsDNA probe to a DNA conjugated reporter probe, wherein said ssDNA annealing region on hybrid dsDNA probe is complementary to a ssDNA annealing region of said DNA conjugated reporter probe such that the two probes are coupled together in a solution;   introducing the hybrid dsDNA probe and DNA conjugated reporter probe solution to a lateral flow assay test strip;   passing the solution through at least one detection zone on said lateral flow assay test strip, wherein said detection zone contains a plurality of embedded target capture probes having a ssDNA region that is complementary to said ssDNA target capture region on said hybrid dsDNA probe;   forming an immobilized complex aggregate comprising said hybrid dsDNA probe, said DNA conjugated reporter probe, and said target capture probe by annealing the complementary target capture region on said hybrid dsDNA probe with the target capture region on said target capture probe;   allowing a plurality of immobilized complex aggregates to form in said detection zone such that a detectable signal is produced.   
     
     
         2 . The method of  claim 1  wherein said bodily fluid sample comprises a saliva sample. 
     
     
         3 . The method of  claim 1  wherein said step of converting comprises the step of converting said RNA transcript biomarker into DNA probe through an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction. 
     
     
         4 . The method of  claim 3  wherein the reagents necessary to produce an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction are pre-loaded into a reaction cylinder. 
     
     
         5 . The method of  claim 1  wherein said dsDNA target sequence is coupled with said ssDNA annealing region and said ssDNA target capture region through a linker. 
     
     
         6 . The method of  claim 5  wherein said linker comprises a tri-carbon chain spacer (C3) linker. 
     
     
         7 . The method of  claim 1  wherein said DNA conjugated reporter probe comprises a conjugated gold nanoparticle (GNP) probe. 
     
     
         8 . The method of  claim 7  wherein said conjugated (GNP) probe comprises a GNP coupled to said ssDNA annealing region through a thiol, PEG 18 , and PolyA construct. 
     
     
         9 . The method of  claim 1  wherein said target capture probe comprises a target capture probe having an immobilized streptavidin base tetramer coupled with a biotin-TEG linker that may further be coupled with said ssDNA target capture probe sequence that is complementary to said target capture region on said hybrid streptavidin. 
     
     
         10 . The method of any of  claims 1  and  8  wherein said lateral flow assay test strip further comprises:
 a conjugate pad in fluid communication with a membrane that allows said solution to flow towards an absorbent pad via capillary action, wherein said absorbent pad is positioned distal to said detection zone. 
 a control zone that may immobilize unbound conjugated gold nanoparticle (GNP) probe 
 
     
     
         11 . The method of  claim 10  wherein said membrane comprises a nitrocellulose membrane. 
     
     
         12 . The method of  claim 1  wherein said RNA transcript biomarker comprises at least one RNA transcript biomarker encoded by at least one nucleotide sequence selected from the group consisting of: SEQ ID NO. 1-444, and 657-865. 
     
     
         13 . A lateral flow assay for the early detection of RNA transcript biomarkers comprising:
 a bodily fluid sample having a host RNA transcript biomarker from a subject;   a reaction cylinder configured to receive the saliva sample and further configured to generate an amplified sample through an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction wherein said amplified sample comprises a hybrid dsDNA probe coupled with a DNA conjugated reporter probe;   a conjugate pad configured to receive the amplified sample;   a membrane in fluid communication with said conjugate pad and further configured to allow said solution to flow through said membrane via capillary action;   a detection zone containing a plurality of embedded target capture probes configured to bind and immobilize said hybrid dsDNA probe;   a control zone configured to bind and immobilize one or more unbound DNA conjugated reporter probes; and   an absorbent pad positioned distal to said detection zone and said control zone.   
     
     
         14 . The lateral flow assay of  claim 13  wherein said bodily fluid sample comprises a saliva sample. 
     
     
         15 . The lateral flow assay of  claim 13  wherein the reagents necessary to produce said isothermal RT-RPA reaction are pre-loaded into said reaction cylinder. 
     
     
         16 . The lateral flow assay of  claim 13  wherein said membrane comprises a nitrocellulose membrane. 
     
     
         17 . The lateral flow assay of  claim 13  wherein said hybrid dsDNA probe comprises:
 a dsDNA target sequence; 
 a ssDNA annealing region; and 
 a ssDNA target capture region. 
 
     
     
         18 . The lateral flow assay of  claim 17  wherein said ssDNA annealing region on hybrid dsDNA probe is complementary to a ssDNA annealing region of said DNA conjugated reporter probe, such that the two probes are coupled together in said amplified solution. 
     
     
         19 . The lateral flow assay of  claim 18  wherein said dsDNA target sequence is coupled with said ssDNA annealing region and said ssDNA target capture region through a linker. 
     
     
         20 . The lateral flow assay of  claim 19  wherein said linker comprises a tri-carbon chain spacer (C 3 ) linker. 
     
     
         21 . The lateral flow assay of  claim 13  wherein said DNA conjugated reporter probe comprises a conjugated gold nanoparticle (GNP) probe. 
     
     
         22 . The lateral flow assay of  claim 21  wherein said conjugated GNP probe comprises a GNP coupled to said ssDNA annealing region through a thiol, PEG 18 , and PolyA construct. 
     
     
         23 . The lateral flow assay of any of  claims 13  and  17  wherein said target capture probes comprise a target capture probe having an immobilized streptavidin base tetramer coupled with a biotin-TEG linker that may further be coupled with said ssDNA target capture probe sequence that is complementary to said target capture region on said hybrid dsDNA probe. 
     
     
         24 . The lateral flow assay of  claim 13  wherein said host RNA transcript biomarker comprises at least one RNA transcript biomarker encoded by at least one nucleotide sequence selected from the group consisting of: SEQ ID NO. 1-444, and 657-865. 
     
     
         25 . A antibody-based lateral flow assay for the early detection of RNA transcript biomarkers comprising:
 a bodily fluid sample having a host RNA transcript biomarker from a subject;   a reaction cylinder configured to receive the saliva sample and further configured to generate an amplified sample through an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction wherein said amplified sample comprises a hybrid dsDNA probe coupled with an antibody conjugated reporter probe;   a conjugate pad configured to receive the amplified sample;   a membrane in fluid communication with said conjugate pad and further configured to allow said amplified sample to flow through said membrane via capillary action;   a detection zone containing a plurality of embedded antibody target capture probes configured to bind and immobilize said hybrid dsDNA probe;   a control zone containing a plurality of embedded antibody target capture probes configured to bind and immobilize said hybrid dsDNA probe;   a capture zone having an antibody configured to bind and immobilize one or more antibody DNA conjugated reporter probes.   
     
     
         26 . The antibody-based lateral flow assay of  claim 25  wherein said bodily fluid sample comprises a saliva sample. 
     
     
         27 . The antibody-based lateral flow assay of  claim 25  wherein the reagents necessary to produce said isothermal RT-RPA reaction are pre-loaded into said reaction cylinder. 
     
     
         28 . The antibody-based lateral flow assay of  claim 25  wherein said membrane comprises a nitrocellulose membrane. 
     
     
         29 . The antibody-based lateral flow assay of  claim 25  wherein said hybrid dsDNA probe comprises:
 a dsDNA target sequence; 
 a 5′ forward ssDNA oligo; and 
 a 5′ reverse ssDNA oligo. 
 
     
     
         30 . The antibody-based lateral flow assay of  claim 29  wherein said 5′ forward ssDNA oligo comprises a 5′ FITC forward oligo. 
     
     
         31 . The antibody-based lateral flow assay of  claim 25  wherein said 5′ reverse ssDNA oligo comprises a 5′ DIG reverse oligo, or a 5′ Biotin reverse oligo. 
     
     
         32 . The antibody-based lateral flow assay of  claim 30  wherein said conjugated reporter probe comprises a gold nanoparticle (GNP) coupled with an antibody forming an antibody conjugated reporter probe. 
     
     
         33 . The antibody-based lateral flow assay of  claim 32  wherein said antibody comprises an anti-FITC antibody. 
     
     
         34 . The antibody-based lateral flow assay of  claims 30  and  33  wherein said FITC antibody binds to said 5′ FITC forward oligo of said hybrid dsDNA probe. 
     
     
         35 . The antibody-based lateral flow assay of  claim 25  wherein said target capture probe of said detection zone comprises an anti-DIG antibody. 
     
     
         36 . The antibody-based lateral flow assay of  claims 31  and  35  wherein said anti-DIG antibody binds to the 5′ DIG reverse oligo of said hybrid dsDNA probe. 
     
     
         37 . The antibody-based lateral flow assay of  claims 25  and  31  wherein said target capture probe of said control zone comprises a target capture probe having an immobilized streptavidin base tetramer coupled with a biotin-TEG linker that may further be coupled with said 5′ Biotin reverse oligo. 
     
     
         38 . The antibody-based lateral flow assay of  claim 30  wherein said target capture probe of said detection zone comprises an anti-rabbit antibody. 
     
     
         39 . The antibody-based lateral flow assay of  claim 25  wherein said host RNA transcript biomarker comprises at least one RNA transcript biomarker encoded by at least one nucleotide sequence selected from the group consisting of: SEQ ID NO. 1-444, and 657-865. 
     
     
         40 . A method of early-pathogen detection comprising the step of:
 collecting a bodily fluid sample from a first subject;   extracting host-derived biomarkers of infection and a pathogen biomarkers from said bodily fluid sample;   quantifying said host-derived biomarkers of infection and a pathogen biomarkers through PCR, real time PCR (RT-PCR), or quantitative real-time polymerase chain reaction (qRT-PCR);   establishing a time-course of the levels of host-derived biomarkers of infection and optionally correlating said host-derived biomarkers of infection with said levels of pathogen biomarkers in said bodily fluid sample;   optionally repeating the four above steps at different time-points;   collecting a bodily fluid sample from a second subject containing a host-derived biomarker of infection;   detecting one or more host-derived biomarkers of infection that correlate to infection with said pathogen.   
     
     
         41 . The method of  claim 40  wherein said bodily fluid sample comprises a saliva sample. 
     
     
         42 . The method of  claim 41  wherein said host-derived biomarkers of infection comprise host-derived RNA biomarkers of infection. 
     
     
         43 . The method of  claim 42  wherein said pathogen biomarkers comprises pathogen biomarkers selected from the group consisting of: viral pathogen biomarkers, bacterial pathogen biomarkers, and pathogen fungal biomarkers. 
     
     
         44 . The method of  claim 43  wherein said viral pathogen biomarkers comprise viral pathogen biomarkers from novel coronavirus SARS-CoV-2. 
     
     
         45 . The method of  claim 40  wherein said viral pathogen biomarkers from novel coronavirus SARS-CoV-2 comprises one or more biomarkers that may be amplified in a PCR reaction by the nucleotide primers according to SEQ ID NOs. 469-480. 
     
     
         46 . The method of  claim 40  wherein said host-derived biomarker of infection comprises host-derived RNA biomarkers of infection and further comprising the step of converting said host-derived RNA biomarkers of infection into a hybrid double stranded DNA (dsDNA) probe through an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction. 
     
     
         47 . The method of  claim 1  wherein said step of detecting comprises the method of  claims 1 - 12 . 
     
     
         48 . A method of detecting an infection in a subject in need thereof, comprising the step of detecting at least one host-derived RNA biomarker of infection from a biological sample provided by said subject, wherein said at least one host-derived RNA biomarker of infection is selected from the group consisting of: a host-derived RNA biomarker of infection encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         49 . The method of  claim 48  wherein said step of detecting comprises the method of  claims 1 - 12 . 
     
     
         50 . The method of  claim 48  wherein said step of detecting comprises the step of detecting said host-derived RNA biomarker of infection comprises detecting a host-derived RNA biomarker of infection using PCR, RT-PCR, or qRT-PCR. 
     
     
         51 . A lateral flow assay configured to detect at least one host-derived RNA biomarker from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker is selected from the group consisting of: a host-derived RNA biomarker encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         52 . An assay configured to detect at least one host-derived RNA biomarker from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker is selected from the group consisting of: a host-derived RNA biomarker encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865, wherein said assay is a PCR assay, RT-PCR assay, or qRT-PCR assay. 
     
     
         53 . A microarray assay configured to detect least one host-derived RNA biomarker from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker is selected from the group consisting of: a host-derived RNA biomarker encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         54 . A lateral flow assay configured to detect at least one host-derived RNA biomarker indicative for a viral infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: IFIT2, ICAM1, ERG1, IFIH1, ISG15, CFB, CXCL10, DDX58, and IRAK2. 
     
     
         55 . An assay configured to detect at least one host-derived RNA biomarker indicative for a viral infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of IFIT2, ICAM1, ERG1, IFIH1, ISG15, CFB, CXCL10, DDX58, and IRAK2, wherein said assay is a PCR assay, RT-PCR assay, or qRT-PCR assay. 
     
     
         55 . A microarray assay configured to detect least one host-derived RNA biomarker indicative for a viral infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: IFIT2, ICAM1, ERG1, IFIH1, ISG15, CFB, CXCL10, DDX58, and IRAK2. 
     
     
         56 . A method of detecting a viral infection in a subject in need thereof, comprising detecting least one host-derived RNA biomarker indicative in a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of IFIT2, ICAM1, ERG1, IFIH1, ISG15, CFB, CXCL10, DDX58, and IRAK2, and said biological sample is saliva. 
     
     
         57 . A lateral flow assay configured to detect at least one host-derived RNA biomarker indicative for a SARS-CoV-2 infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: MX1, PARP12, IFITM2, CD68, and SERINB3. 
     
     
         58 . An assay configured to detect at least one host-derived RNA biomarker indicative for a SARS-CoV-2 infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of MX1, PARP12, IFITM2, CD68, and SERINB3, wherein said assay is a PCR assay, RT-PCR assay, or qRT-PCR assay. 
     
     
         59 . A microarray assay configured to detect least one host-derived RNA biomarker indicative for a SARS-CoV-2 infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: MX1, PARP12, IFITM2, CD68, and SERINB3. 
     
     
         60 . A method of detecting a SARS-CoV-2 infection in a subject in need thereof, comprising detecting least one host-derived RNA biomarker in a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker is indicative for a SARS-CoV-2 infection is selected from the group consisting of MX1, PARP12, IFITM2, CD68, and SERINB3, and said biological sample is saliva. 
     
     
         61 . A lateral flow assay configured to detect at least one host-derived RNA biomarker indicative for an influenza infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: PLRG1, MSC, NKG7, NME8, and MMP12. 
     
     
         62 . An assay configured to detect at least one host-derived RNA biomarker indicative for an influenza infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of PLRG1, MSC, NKG7, NME8, and MMP12, wherein said assay is a PCR assay, RT-PCR assay, or qRT-PCR assay. 
     
     
         63 . A microarray assay configured to detect least one host-derived RNA biomarker indicative for an influenza infection from a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker indicative for a viral infection is selected from the group consisting of: PLRG1, MSC, NKG7, NME8, and MMP12. 
     
     
         64 . A method of detecting an influenza infection in a subject in need thereof, comprising detecting least one host-derived RNA biomarker in a biological sample provided by a subject, wherein said at least one host-derived RNA biomarker is indicative for an influenza infection is selected from the group consisting of PLRG1, MSC, NKG7, NME8, and MMP12, and said biological sample is saliva. 
     
     
         65 . The method of any of  claims 51 - 64 , wherein said RNA biomarker is selected from the group consisting of: a host-derived RNA biomarker encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         66 . A nucleotide sequence encoding a host-derived RNA biomarker used to detect an infection in a subjected in need thereof, wherein said RNA biomarker is selected from the group consisting of: a nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         67 . A method of detecting a host-derived RNA biomarker comprising:
 collecting a bodily fluid sample potentially containing a host-derived RNA biomarker and optionally a biomarker of a viral, bacterial, or fungal infection;   identifying a transcript of said host-derived RNA biomarker in the sample, and optionally a biomarker of a viral, bacterial, or fungal infection using a method selected from the group consisting of: PCR, RT-PCR, qPCR, transcript sequencing, a lateral flow assay, hybridization assay, microarray, nucleic acid detection assay.   
     
     
         68 . The method of  claim 67 , wherein said bodily fluid sample comprises a saliva sample. 
     
     
         69 . The method of  claim 68 , wherein said host-derived biomarkers of infection comprise host-derived RNA biomarkers of infection. 
     
     
         70 . The method of  claim 69 , wherein said host-derived RNA biomarkers of infection comprises pathogen biomarkers selected from the group consisting of: viral pathogen biomarkers, bacterial pathogen biomarkers, and pathogen fungal biomarkers. 
     
     
         71 . The method of  claim 70 , wherein said viral pathogen biomarkers comprise viral pathogen biomarkers from novel coronavirus SARS-CoV-2. 
     
     
         72 . The method of  claim 71 , wherein said viral pathogen biomarkers from novel coronavirus SARS-CoV-2 comprises one or more biomarkers that may be amplified in a PCR reaction by the nucleotide primers according to SEQ ID NOs. 469-480. 
     
     
         73 . The method of  claim 69 , wherein said host-derived biomarker of infection comprises host-derived RNA biomarkers of infection and further comprising the step of converting said host-derived RNA biomarkers of infection into a hybrid double stranded DNA (dsDNA) probe through an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) reaction. 
     
     
         74 . The method of  claim 69 , wherein said host-derived biomarker of infection comprises a host-derived RNA biomarker of infection is selected from the group consisting of: a host-derived RNA biomarker of infection encoded by the nucleotide sequence according to SEQ ID NOs. 1-444, and 657-865. 
     
     
         75 . The method of  claim 69 , wherein said biomarker of a viral, bacterial, or fungal infection comprises an RNA biomarker of a viral, bacterial, or fungal infection.